SUMMARY This renewal builds on discoveries driving the current R37 program that established epigenetic mechanisms regulating osteoblast differentiation. Epigenetic mechanisms that include various modifications of DNA structure have emerged as key regulators of cell phenotypes. By examining eight different histone modifications during four stages of osteoblastogenesis from mouse bone marrow stem cells (MSCs), we characterized for the first time a ?signature? of specific histone modifications that are associated with dynamic changes in gene expression during the temporal progression of osteogenesis. These histone modifications also predicted ?enhancers?, which are critical cis-regulatory elements that contribute to local gene expression, and ?super enhancer? domains (SEDs) that include regulatory elements for multiple transcription factors (e.g., Runx2, Sp7, homeodomain proteins). SEDs function in chromatin organization via long range intra- and inter- chromosomal interactions that coordinate control of gene cohorts responsible for lineage specification and distinct cell identity. Further, by overlapping our ChIP-Seq data sets from Runx2 and the chromatin-organizing protein CTCF with SEDs, we identified a subset of SEDs that we now propose are putative ?bone-essential super-enhancers? and candidates for the important decision stage of commitment to osteogenesis from MSCs. Our working hypothesis is that a small cohort of SEDs with binding sites for multiple bone related transcription factors establish and sustain the osteoblast phenotype through coordinate regulation of gene cohorts and at the higher level of chromatin organization. We will in: Aim1- analyze the functional effects of SEDs on osteoblastogenesis through directed inhibition and activation of SED activity using CRISPR/Cas9 in MSCs; Aim 2- determine the chromosomal domains that interact with SEDs to control multiple genes and networks that commit MSCs to the osteogenic phenotype through chromatin organization; and Aim 3- demonstrate in vivo by SED activation in MSCs (using CRISPRa), stimulation of bone formation for translational applications in pre-clinical mouse models. Impact: These studies are pioneering a new level of gene regulation for MSC lineage commitment to osteogenesis, based on an emerging understanding of SED functions in other tissues. By identifying and characterizing SEDs in MSCs, we will discover multi-dimensional transcriptional hubs and protein complexes that activate networks responsible for establishing the phenotype of osteoblast populations. Importantly, knowledge of the chromatin organization that stabilizes the osteogenic phenotype impacts on future novel treatment strategies for skeletal disorders.